Esercitazione 2 maggio pomeriggio - Unifem.docente.unife.it/francesco.bernardi/materiale... ·...
Transcript of Esercitazione 2 maggio pomeriggio - Unifem.docente.unife.it/francesco.bernardi/materiale... ·...
![Page 1: Esercitazione 2 maggio pomeriggio - Unifem.docente.unife.it/francesco.bernardi/materiale... · 0-1-2-3-4 mismatches + next to PAM 0-0-3-7-64 0-0-0-0-1 74 off-targets Genome Browser](https://reader036.fdocuments.in/reader036/viewer/2022071023/5fd8561235ce2b2a0647dd82/html5/thumbnails/1.jpg)
4. Selection of target sequence/ gRNA design:Plenty of webtools available:
• Predicted- on-target activity
• Predicted off-target activity
• … much more
http://crispor.tefor.net/
Selecting gRNA
https://omictools.com/genome-editing-category
![Page 2: Esercitazione 2 maggio pomeriggio - Unifem.docente.unife.it/francesco.bernardi/materiale... · 0-1-2-3-4 mismatches + next to PAM 0-0-3-7-64 0-0-0-0-1 74 off-targets Genome Browser](https://reader036.fdocuments.in/reader036/viewer/2022071023/5fd8561235ce2b2a0647dd82/html5/thumbnails/2.jpg)
Selecting gRNA4. Selection of target sequence/ gRNA design:Plenty of webtools available:
• Predicted- on-target activity
• Predicted off-target activity
• … much more
![Page 3: Esercitazione 2 maggio pomeriggio - Unifem.docente.unife.it/francesco.bernardi/materiale... · 0-1-2-3-4 mismatches + next to PAM 0-0-3-7-64 0-0-0-0-1 74 off-targets Genome Browser](https://reader036.fdocuments.in/reader036/viewer/2022071023/5fd8561235ce2b2a0647dd82/html5/thumbnails/3.jpg)
Selecting gRNA4. Selection of target sequence/ gRNA design:Plenty of webtools available:
• Predicted- on-target activity
• Predicted off-target activity
• … much more
![Page 4: Esercitazione 2 maggio pomeriggio - Unifem.docente.unife.it/francesco.bernardi/materiale... · 0-1-2-3-4 mismatches + next to PAM 0-0-3-7-64 0-0-0-0-1 74 off-targets Genome Browser](https://reader036.fdocuments.in/reader036/viewer/2022071023/5fd8561235ce2b2a0647dd82/html5/thumbnails/4.jpg)
Measuring Cas9 Efficacy
NON-HOMOLOGOUS END JOINING (NHEJ)
repairs with indels
How to measure the DSB efficacy of a CRISPR sgRNA and the nature of the mutations?
![Page 5: Esercitazione 2 maggio pomeriggio - Unifem.docente.unife.it/francesco.bernardi/materiale... · 0-1-2-3-4 mismatches + next to PAM 0-0-3-7-64 0-0-0-0-1 74 off-targets Genome Browser](https://reader036.fdocuments.in/reader036/viewer/2022071023/5fd8561235ce2b2a0647dd82/html5/thumbnails/5.jpg)
1. Sequencing based techniques
2. Denaturation based techniques
3. Alternative techniques
Measuring Cas9 Efficacy
![Page 6: Esercitazione 2 maggio pomeriggio - Unifem.docente.unife.it/francesco.bernardi/materiale... · 0-1-2-3-4 mismatches + next to PAM 0-0-3-7-64 0-0-0-0-1 74 off-targets Genome Browser](https://reader036.fdocuments.in/reader036/viewer/2022071023/5fd8561235ce2b2a0647dd82/html5/thumbnails/6.jpg)
1. Sequencing based techniques
Measuring Cas9 Efficacy
From Gelmini et al., Trends Biotechnol 2017
![Page 7: Esercitazione 2 maggio pomeriggio - Unifem.docente.unife.it/francesco.bernardi/materiale... · 0-1-2-3-4 mismatches + next to PAM 0-0-3-7-64 0-0-0-0-1 74 off-targets Genome Browser](https://reader036.fdocuments.in/reader036/viewer/2022071023/5fd8561235ce2b2a0647dd82/html5/thumbnails/7.jpg)
1. Sequencing based techniques: TIDE
Measuring Cas9 Efficacy
Tracking of Indels by DEcomposition
http://tide.nki.nl
Eva K. Brinkman, Tao Chen, Mario Amendola, Bas van Steensel
35%
32%
18%
15%
35%32%
15%18%
-4-3-2-10+2+1=
+
+
+
0
-1
-4
+1
R2 = 0.99
+3